Eukaryotic cells can shift from a proliferating state to a quiescent state only during a brief window of the cell cycle. Temin, J. Cell. Phys. 78:161 (1971). Thus, depending on their position in the cell cycle, cells deprived of mitogens such as those present in serum when mammalian cells are examined, will undergo immediate cell cycle arrest, or they will complete mitosis and arrest in the next cell cycle. The transition from mitogen-dependence to mitogen-independence occurs in the mid- to late-G1 phase of the cell cycle. Pardee, Proc. Natl. Acad. Sci. USA 71:1286 (1974), showed that many different anti-mitogenic signals cause the cell cycle to arrest at a kinetically common point, and further showed that the cell cycle becomes unresponsive to all of these signals at approximately the same time in mid- to late-G1. This point was named the restriction point, or R point.
Time-lapse cinematography of mitotically proliferating single cells has also been used to precisely map the timing of the cell cycle transition to mitogen-independence. This confirmed that mitogen depletion or other growth inhibitory signals cause post-mitotic, early-G1 cells to immediately exit the cell cycle, and that cell cycle commitment (autonomy from mitogenic signals), occurs in mid-G1 (Larsson et al., J. Cell. Phys. 139:477 (1989), and Zetterberg et al., Proc. Natl. Acad. Sci. USA 82:5365 (1985)). Together these observations (1985)). Together these observations show that the mitogen-dependent controls on cell proliferation are linked to cell cycle progression.
Transit through G1 and entry into S phase requires the action of cyclin-dependent kinases (Cdks) (Sherr, Cell 79:551 (1994)). Growth inhibitory signals have been shown to prevent activation of these Cdks during G1 (Serrano et al., Nature 366:704 (1993); Hannon and Beach, Nature 371:257 (1994); El-Deiry et al., Cell 75:89 (1993); Xiong et al., Nature 366:701 (1993); Polyak et al., Cell 78:59 (1994); Toyashima and Hunter, ibid., p. 67; Lee et al., Genes & Dev. 9:639 (1995); Matsuoka et al., ibid., p. 650; Koff et al., Science 260:536 (1993)). The catalytic activity of Cdks is known to be regulated by two general mechanisms, protein phosphorylation and association with regulatory subunits (Gould et al., EMBO J. 10:3297 (1991); Solomon et al., ibid., 12:3133 (1993); Solomon et al., Mol. Biol. Cell 3:13 (1992); Jeffrey et al., Nature 376:313 (1995); Morgan, Nature 374:131 (1995)). Among the regulatory subunits, the association of Cdks with inhibitory CKI subunits (Cyclin-dependent Kinase Inhibitors) has been most closely correlated with the effect of mitogen depletion on cell proliferation and Cdk activity.
Plant cells were used in early studies of cell growth and division to establish the phases of the eukaryotic cell cycle. (Howard et al., Heredity 6(suppl.):216–273 (1953)), but little is known about the molecular mechanisms of plant cell cycle regulation. Plant cells that cease dividing in vivo due to dormancy, or in vitro due to nutrient starvation, arrest at principal control points in G1 and G2. (van't Hof et al., in The Dynamics of Meristem Cell Populations, Miller et al. eds., Plenum, New York, pp 15–32 (1972), Gould et al., Protoplasma 106:1–13 (1981)). Generally, this pattern is in agreement with that seen in other eukaryotic systems. Homologues of cdc2 kinase have been isolated from a number of plant species, including pea (Feller et al., Proc. Natl. Acad. ScL USA 87:5397–5401 (1990)), alfalfa (Hirt et al., Proc. Natl. Acad Sci USA 88:1636–1640 (1991) and Hirt et al., Plant J. 4:61–69 (1993)), and from A. thaliana. (Ferreira et al., Plant Cell 3:531–540 (1991), Hirayama et al., Gene 105:159–165 (1991)), among others. Also, a number of cDNA sequences encoding plant cyclins with A-, B- or D-type characteristics or having mixed A- and B-type features have been isolated from various species, including carrot and soybean (Hata et al., EMBO J. 10:2681–2688 (1991)), and Arabidopsis (Hemerly et al., Proc. Natl. Acad. Sci. USA 89:3295–3299 (1992), and Soni et al., Plant Cell 7:85–103 (1995)), among others.
Recently DNA sequences encoding plant-cyclin dependent kinase inhibitors of Arabidopsis have been identified (WO 99/14331; Wang et al., Plant J. 15:501–510 (1998); each incorporated herein by reference). The proteins encoded by the DNA sequences have been suggested modulators of plant cell division because of their binding to cyclins and similar in vitro inhibitory effects on kinases. It has also been suggested that partial and/or total elimination of a gene or reducing the expression of a gene encoding a plant cyclin-dependent kinase inhibitor could influence and would likely inhibit cell division (WO 99/14331).
Genetic engineering of plants, which entails the isolation and manipulation of genetic material, and the subsequent introduction of that material into a plant, plant tissue, or plant cells, has changed plant breeding and agriculture considerably over recent years. Increased crop food values, higher yields, feed value, reduced production costs, pest resistance, stress tolerance, drought resistance, and the production of pharmaceuticals and biological molecules as well as other beneficial traits are all potentially achievable through genetic engineering techniques.
The ability to manipulate gene expression provides a means of producing new characteristics in transformed plants. For example, the ability to increase the size of a plant's root system would permit the increased nutrient assimilation from the soil. Moreover, the ability to increase leaf growth, i.e., an increase in leaf size and/or leaf number, would increase the capacity of a plant to assimilate solar energy. Obviously, the ability to control the growth of an entire plant, or specific target organs of a plant would be very desirable.
Cell cycle control genes can be employed to improve growth and development in the economically valuable portions of crop plants, including both dicotyledonous plants and monocotyledonous plants. In monocotyledons the additional use of cell cycle control genes or protein can be useful to improve their regenerability from callus.
Further, cell cycle control genes are potential sites to influence cell division and behavior at stages of plant development when cell number influences the final yield of economically valuable tissue. A specific example is the number of rounds of nuclear division at the multinucleate stage of endosperm development in cereal grains, or at the stage of fruit or flower development.
Based on the foregoing, it is clear that a need exists for methods for modulating the cell division of plant cells, plant tissues, and plants harboring one or more functionally inactivated endogenous cyclin inhibitor genes, and optionally also harboring a transgene encoding a heterologous cyclin inhibitor polypeptide or mutant variant cyclin inhibitor polypeptide which is expressed in at least a subset of host cells. Thus, it is an object of the invention herein to provide methods and compositions for increasing cell division by functionally inhibiting the expression or activity of plant cyclin inhibitors.
Further, the present invention provides for nucleic acid sequences encoding the plant D-like cyclin binding protein BRO4. Also, the present invention provides targeting transgenes to inactivate an endogenous cyclin inhibitor gene, particularly the genes which encode proteins having binding motifs for plant D-like cyclin and cyclin dependent kinases. It is also an object of the invention to provide methods to produce transgenic plant cells, plant tissues, and transgenic plants harboring a correctly targeted transgene of the invention. The methods may also be used to inactivate cyclin inhibitor genes in cells explanted from a plant (e.g., for ex vivo insertion), such as to impart to the resultant targeted cells a phenotype which results from an increased cell proliferation phenotype.